Cell division cycles and checkpoints are altered during both development and cancer. In this proposal, we focus on a developmental cell cycle variation called the endocycle (G/S cycle), which results in polyploid cells. Evidence suggests that an inappropriate switch into an endocycle contributes to genome instability and cancer. There is a large knowledge gap, however, in understanding how this alternative cell cycle is regulated and how it compromises genome integrity. Our overall objective is to define mechanisms in endocycling cells that repress apoptosis and contribute to aneuploidy when these cells return to mitosis. To achieve this goal, our proposal leverages the complementary expertise of the Calvi and Walczak labs in Drosophila endocycles and human cell mitosis respectively. Our central hypothesis is that a specific remodeling of the cell cycle transcriptome promotes endocycles, represses apoptosis, and causes genome instability. This hypothesis emanates from our discovery that endocycling cells in Drosophila development repress apoptosis by blocking the p53 genotoxic stress pathway. Experimental ablation of mitosis in Drosophila creates induced endocycling cells (iECs), and is sufficient to repress apoptosis. This suggests that there is an unsuspected link between cell cycle programs and apoptotic pathways. iECs can later resume mitotic divisions that are error prone, resulting in proliferative aneuploid daughter cells. We are also evaluating conservation to humans. Ablation of mitosis also creates human polyploid iECs, which can return to a mitosis that is severely error prone. Our transcriptome analysis has provided crucial insights into mechanism and leads to a unifying hypothesis for how endocycle regulation is linked to the repression of apoptosis and genome instability. Our central hypothesis will be tested in three specific aims: In Aim 1, we will define the molecular mechanisms by which the cell cycle and apoptotic pathways are linked in Drosophila. A novel unbiased genetic screen also exploits the power of Drosophila to discover new players in this process. Aim 2 uses molecular and genetic methods to test the hypothesis that persistent replication stress in Drosophila iECs compromises genome integrity. Aim 3 takes advantage of our expertise in human cell mitosis and advanced cellular imaging to define the mechanisms of chromosome segregation errors when human iECs return to mitosis. We also test the hypothesis that a specific remodeling of the cell cycle contributes to these errors, spawning proliferative, aneuploid daughter cells. The outcome of these integrated aims will show how endocycling cells avoid apoptosis despite persistent replication stress, and how this stress, with errors in mitosis, generates aneuploid cells. A transient switch to an endocycle may contribute to therapy resistance of the cancer cell, with a return to mitosis generating high rates of aneuploidy that contributes to disease progression. This research is significant because the outcomes will define new mechanisms for cell survival and aneuploidy, and will ultimately lead to more effective cancer therapies.